JPS60252464A - Substrates for hydrolase and manufacture - Google Patents

Abstract

Chromogenic substrates of the formula A-O-B are described, in which A represents the radical in the ion A-O- of a compound A-OH, whose absorbance is measured photometrically after hydrolysis of A-O-B, while B is the radical in a compound B-OH which makes the compound specific for the reaction with a given enzyme, and where A-OH is a hydroxynitropyridine, a hydroxynitrobenzopyranone, a dibenzox(or thi)azine or a dibenzopyranone or a derivative thereof, and B is a sugar or sugar derivative, an amino acid or an oligopeptide or an inorganic acid. These compounds can be used for the detection and determination of hydrolases.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to substrates for hydrolase measurements, processes for their preparation, their use in hydrolase measurements and medicaments suitable for that purpose.

Detection or measurement of hydrolases is important for the diagnosis and progress monitoring of various diseases. For example, amylase measurement is useful for pancreatic diagnosis, and acid phosphatase is useful for prostate cancer diagnosis.
J. Haschen)'s "Enzyme Diagnostics"
J, Guetav Fisch
θr) Publishing, Stuttgart, 1970], esterases as indicators of leukocytes (European Patent Application EP-A-12,957) or phosphatases or galactosidases as indicators for immunoassays in enzyme immunoassays. It is important in

The determination of hydrolases with a pH optimum below 7 is currently generally carried out photometrically using two-point measurements.

Two-point measurements generally require more time than measurements that can be carried out kinetically and, moreover, cannot be used for widely used automated analysis, so only expensive manual measurements are possible. Furthermore, they are less accurate and difficult to inspect than kinetic methods, which can lead to erroneous results.

A few kinetically performable measurements of hydrolases with an acidic pH optimum are described, for example, in West German Patent No. 2,1
No. 15,748 for acid phosphatase, or require a several-step reaction, e.g.
Fluorescent chromogenic substrates such as f'luoro-genic substrates
). However, several-step reactions can lead to interference with the measurement due to impurities in the reagents, especially in the coenzymes, and competitive reactions with components of the biological fluid. Furthermore, this method increases the price. Fluorescent substances can only be measured with special equipment, are difficult to standardize and are subject to interference under various influences, which leads to low accuracy and erroneous results. Moreover, fluorescent chromogenic substrates cannot be used to measure enzyme activity at the saturating concentrations conventionally required, since under these conditions the fluorescence of the reaction product is considerably quenched by absorption of the substrate. Laborious correction and standardization operations may be necessary to obtain adequate results.

p-Nitrophenyl-α-malt-y' key: For example, testomer/l/(Tθ daily mar) [F]-
Amylase, Behringwerke
ke) A method for measuring α-amylase activity using AG, Federal Republic of Germany, is already known, in which the measurement is carried out kinetically at pH 7, which means that the absorbance of the liberated nitrophenol is It has five drawbacks: it is strongly value dependent and the test is susceptible to interference. Furthermore, about 509b of sensitivity is lost when the nitrophenyl group is the chromogen, since the molar absorptivity of nitrophenol is p
This is because, for example, at H7, the size is only half that at pH9. However, at pH 9 it is not possible to measure the activity of amylase, since it loses its activity under this condition.

Furthermore, all previous kinetically possible amylase measurements (DE 275.201,
No. 2,751,372, No. 2,822,364, No. 3,000,292 and Japanese Patent No. 11 to 15
(see No. 8) is a several-step reaction requiring one or more coenzymes. This is associated with the disadvantages already mentioned above.

The sensitivity of the detection system is of critical importance in the measurement of trace components in biological fluids. Therefore, enzyme immunoassays have been developed in addition to radioimmunoassays in recent years. It allows the detection of trace amounts of proteins in human serum using, for example, galactosidase in an indicator reaction. However, all previous methods have the disadvantage of requiring culture times ranging from one to several hours. With greater sensitivity of the indicator reaction, the incubation time can be reduced, or with the same incubation time the sensitivity can be correspondingly increased. Esterase is a diagnostic parameter for the presence of leukocytes. Nowadays, in addition to specificity, a high degree of sensitivity is also required in order for the results to be readable already after a reaction time of 2-3 minutes. Visual test systems, such as test strips, require dyes that produce the optimal color display for the human eye (red, green, blue). The yellow color of nitrophenol or nido, loaniline is very disadvantageous in this respect. The achievable sensitivity therefore depends critically on the chromogenic molecular portion of the substrate.

The chromogenic substrates of the current state of the art are not suitable for visual determination because of the color they produce (yellow) or because of their low reactivity. Thus, for example, European patent no.
No. 07,407 (sulfophthalene), No. 0.008,
No. 438 (Azo dye), No. 0.012,957 (Indoxyl) or West German Patent No. 3.0 [15,84]
Using the chromogenic amino acid ester described in No. 5 (Indoaniline), the lower limit of white blood cells is 2 within about 2 minutes required for color development.
A sensitivity of ~5/μt is obtained. However, much closer limits than this are desirable for diagnosis. In this substrate, the sensitivity can be increased by additionally adding promoters.

US Patent No. 3.57F! , No. 465 describes the fluorescence of ester 2-ase substrates with chromogens of the indoxyl and resorufin type, which, however, can only be used in the determination with suitable equipment (fluorescent needles). There is an interesting statement in column 5, lines 61-65 of this patent that the corresponding indoxyl derivatives are degraded much faster than the resorufin derivatives. Therefore, in connection with the above, it is expected that resorufin esters will also have even lower sensitivity in the detection of esterases, for example from leukocytes.

Another drawback of the substrate described in said patent is its reactivity towards other esterases. Esterases, such as cholinesterase, acylase, lipase or phosphatase, as well as proteases, such as chymotrypsin, react with resorufin acetate or butyrate (see Table 3 of the above-mentioned US patent).

Therefore, in order for the kinetics of hydrolases to be able to be determined, which can be carried out using automatic analyzers, or for the indicator reactions to be sensitive for enzyme immunoassays to be constructed, it is necessary to have as low a pKa value as possible and as high an anion as possible. Obtain an indicator sensitive for enzymatic reactions in which an anionic dye (dye is here understood as a compound having an absorption in 3D [J ~ 760 μm]) with a molar absorptivity of is liberated by enzymatic hydrolysis. Furthermore, there has been a problem in finding enzyme substrates that react specifically with only one type of enzyme and thus allow specific determination of enzymes or groups of enzymes in enzyme mixtures such as those present in biological fluids. There was nM to obtain .

Surprisingly, it has now been found that a heterocyclic compound A-
Representation is the residue of the ion A-0'' of OH, while B is the residue of the compound B-OH which makes that compound specific for reaction with certain enzymes, and where A-OH is a , general formula ■ 1:1 large (in the formula, R1 is 0H1NO2 or halogen and R3,
R4 and R5) can be i or No2, with up to 2 NO2 being present), b, a compound having the general formula ■3, in which B1 is H or 1 to 12, preferably 1 to 3 alkyl having 4 carbon atoms and R2, R3 and R4
can be H or NO2, in which case 1 or 2 NO2 are present); (
CH3)2 and R3 can be O or S), or d, of the general formula ■ R 噌 (wherein B1 is aryl, preferably phenyl, CN or 1 to 12, preferably 1 to 5 alkyl having a carbon atom), wherein B-OH is a sugar or sugar derivative, in particular glucose, maltodextrin, gunctose, glucuronic acid, N-acetylglucosamine or fucose, an amino acid or an oligonucleotide. Peptides, each of which may carry a protecting group, in particular a toluenesulfonyl group or a carbobenzyloxy (CBZ) group, in particular N-tosylalanine or CBZ-glycine, or an inorganic acid, in particular phosphoric acid or sulfuric acid] It was found to be a substrate with high detection sensitivity for hydrolase measurements.

The invention also relates to compounds having the formula A-0-B and the definitions given above, to processes for their preparation and to their use in hydrolase measurements.

For kinetic measurements of hydro2-ases having an optimum pH of 7 or less, substrates whose hydrolysis liberates a dye with a pKa value of 7 or less are particularly suitable. The dyes used exhibit an intense color already at pH 7 or below and can be detected photometrically or with corresponding sensitivity (by the naked eye).

There has been prejudice against the possibility of synthesizing glycosides with aglycones having low pKa values, ie relatively strongly acidic aglycones, since a low thermodynamic stability is expected for such glycosides.

Such compounds are known to tend to have anhydrous properties that are thermodynamically considerably more unstable than conventional glycosides and esters.

Surprisingly, however, the derivatives of the above-mentioned heterocycles with low pKa values are similar to the homocyclic compounds hitherto used in the synthesis of glycosides and esters, such as 2,
It was found to have sufficient kinetic stability to be obtained in satisfactory yields compared to 4-dinitrophenylgalactoside.

Another surprising property of these compounds is that they have relatively sharp, in some cases very high and long wavelength absorption bands, which makes them useful as indicators for widely used absorption photometry measurements. It is specially adapted as

Moreover, even if the pigment components have no similarity to natural analogous substrate components (aglycone moieties in disaccharides or starches, alcohol components in esters), the above-mentioned compounds can be used as their substrates. It is true that it is particularly rapidly degraded by hydrolases.

This was not expected.

A substrate with the general formula A-0-B is prepared in three steps.

(1) Preparation of the AOH moiety, i.e., the dye; (2) B
(3) Preparation of substrates A, -0-B from AOH and BOH by methods that yield substrates with the desired stereoisomerism, especially in carbohydrates and carbohydrate derivatives.

The known dyes can be prepared by the following method: nitropyridine derivatives and coumarin derivatives are generally prepared by nitration of the corresponding starting materials by dropwise addition of a HNo5/a2so4 mixture in sulfuric acid solution at about D°C. [Sh, ah) and Mehta
ta)'s ``Jie Intian Kem Tsuk (J,
Engineering Chem, 8oc,) J (1954)
, p. 784, Bechman and Obermul'1er, vol. 34 of Bar-J (1901, no. 6).
Page 60, Chakrawarti
) by Mr.
dian Chem, Soc, )Ber (Ber -)j
Volume 34 (19 (11 years), page 660, Mr. Shakrawartei's [J, Eng.
diaHChem, Soc, ) J Vol. 12 (1955), pp. 791-795, < ) Burton et al. [J, Chem.

Soc, Perin Month If (1972) No. 1953-
1958, Brignell et al. [J, Chem, Soc, B (1968), p. 1477,
e Selems)'s ``J,
Org, Chem, ) J Volume 33 (1968) No. 4
Page 78, Talik and Ta'L
ik), “Rocziniki Chemie”
Chemii), Antsk Chem Boronorum (A
nn, Soc, Chem・P010nOrum Month 40th
(1966), p. 1187], Resazurin (3)
10- of H-7-hydroxyphenoxazin-3-one
Oxide) is described by Niθtzki et al.
Ber, clarer
Deut, Chem, ()es, )J Volume 22 (188
9) According to the description on page 5020, Resorufin C3F
i-7-hydroxyphenoxazin-3-one) is produced by reducing resazurin with sodium borohydride.
6-hydroxy-9-phenyl-3H--? Santen-5-one is described by Mr. Nathen in J. Amer, Chem, Soc, Vol. 47 (1925), p. 1079, and other phenoxazines and xanthenones. "Collection Czechoslovakim Commun" by Mr. Stuzka et al.
Hoslov, Chem, Coamun, ) J, Vol. 64 (1969), p.
ehrman) and de Gottlau (dθGott
rau)'s ``Ber Monthly Volume 58, Page 2575 (
(1905).

In the following, cyanorufin
6H-6-hydroxy-9-cyanoxanthin-6-one, which is expressed as This was prepared by the method described in the Examples.

Also new is 7-hydroxy-3-nitrocoumarin, which was prepared by ring closure of nitrostyrene derivatives. It has a melting point of 192-198 °C and a maximum absorption of 45 D nm above pH 8, and a
Below 6, the maximum absorption exists at 390 nm.

Carbohydrate B, optionally carrying a protecting group, suitable for the preparation of compounds with formula A-0-B by reaction with A-OH
Known derivatives of -OH are [Methoasin Ca
rbohydrate Chemistry) J Volume ■, Academic Press
) Publishing, 1963. Amino acids or oligopeptides with protecting groups can be purchased or purchased from Example 16 [J.
amer, Chem, Soc, ) J Volume 59 (1937
1116-1118].

New compounds having the formula A-0-B can be prepared by methods known per se from compounds having the formula A-OH and 2B-OH.
prepared from reactive derivatives of compounds with

Compounds with formula A-0-B can be used as substrates for the detection and measurement of hydrolases.

The dye A-OH liberated upon decomposition has considerable advantages compared to the state of the art. The pKa value of the often used dye p-nitrophenol, i.e. the pH value at which half of the p-nitrophenol exists in dissociated form as the 7 enolate anion, is 7, whereas the pKa value of the dye A-OH used in the present invention is 7. The pKa value is less than 6 (see Table 1). This means that in the pH range below 7 there is already a considerably higher absorption than p-nitrophenol at the same molar concentration;
This therefore means that freshly prepared substrates from this dye may be measured with considerably more sensitivity.

The advantages of using the new substrate compared to the state of the art are described below by way of example.

The activity of α-glucosidase in human urine was determined by the use of a novel substrate, 2H-7-0-(α-D-gelcopi-2-nosyl)-4-me? 8-Nitrobenzopyran-2-one (compound S1, see Example 1) can be determined with great sensitivity in kinetic tests. Since the optimum pH of α-glucosidase is 4 to 6, the activity cannot be measured kinetically using the conventional substrate p-nitrophenyl-α-glucopyranoside. This is because phenol exhibits very little absorption at 405 nm (see Table 1).

Table 1 Selected as an example at different pH values 4-me IF-8-nitrobensohyran-2-one 4.45 3-Hydroxy-2-nitropyridine 5. Lusorufin 5.95 3-hydroxy-4-nitro-2-chloropyridine 513-hydroxy-2-nitro-6-chloropyridine
3.9 p-Nitrophenol 7.0 0-Nitrophenol 108 a] Calculated value b) Variable wavelength photometer C] Absorbance wave of 7-scaled dye for mercury lamp and fixed wavelength 36 g 5.80 11 .3 1 6.10 1
6.25 1 /l+, 30405 0.502.32
5.09 5.80 5.90b) 578 0.75 5.7 22.80 39.6 4
1.000s72)? ! 2%, '6449.8
755.42405 4.93 5.34 5.40
5.48 5.4365 7:84 11.87i2.
9 13.2 13.2405 Q, 20 α40 1
．． 50 9.7 1&30405 CL47 0.48
0.75 2.34 Photometer with 4.01 Ilter The test mixture with this substrate is therefore made alkaline with additives such as bicarbonate in order to reach a measurable absorbance after a given reaction time. Must be. , - In contrast, an absorption difference of 0.040 per minute can be determined in normal human urine with compound S1 as substrate in a kinetic test, thus making it possible to automate the measurement.

It is known that inhibition and quality that affect the activity of glucosidase appear in the urine. In that case, the inhibitor must be removed by suitable measures, such as dialysis or C/I/filtration. The use of purified β-glucosidase shows that substrate S1 is a specific substrate for α-glucosidase, since it
This is because it is not degraded by glucosidase.

Another possibility to measure α-glucosidase activity kinetically is 3H-7-0-(α-glucopyranosyl)-
The use of phenoxazin-3-one (resorufin-α-D-glucopyranoside). pKa of resorufin
It is true that the value is at 5.9, however 57
For example]) H6 due to its very high extinction at 8 nm
urinary glucosidase can be measured with sufficient sensitivity. When run at 37°C, 400 μL of reagent and 200 μL of urine
An absorption difference of 0.136 per minute was observed using μt. 57
Measurement at 8 nm had the advantage of not being interfered with by colored components in urine. Again, the use of β-glucosidase means that resorufin-α-glucopyranoside is
It was shown to be a specific substrate for glucosidase.

Determination of β-glucosidase activity in human urine has not previously been possible using conventional tests due to its low sensitivity.

β-Glucosidase activity was measured comparatively using 3-0-(β-D-glucopyranosyl)-3-hydroxy-2-nitroviridine (compound s5) and p-nitrophenyl-β-D-glucopyranoside. The results are shown in Table 2 and using the new substrate S5 about 7
It is proven that a twice as large absorption change is observed.

Table 2 Measurement of β-glucosidase activity in urine samples Absorption difference 7 molecules -25°C p-nitrophenyl derivative 0.047685 (13
52 To measure α-amylase, the following new substances,
That is, 2H-7-0-(α-D-maltotriosyl)
-4-methyl-8-nitrobenzopyran-2-one (compound El j O), the corresponding maltotetraosyl compound (compound 511)
Comparative measurements were made using -maltotriocytate.

Approximately 14 times greater absorption changes were measured with substrate E111 than with the nitrophenyl derivative and even 32 times with substrate 810 (see Table 3).

Table 3 Measurement of α-amylase activity in urine samples without using the coenzyme α-glucosidase Nitrophenyl) L/visitor 0.020 days 11 Q, 1
34 s10 0.635 This high absorption change with the new substrate is achieved without the participation of α-glucosidase as a coenzyme, which is necessary in conventional test systems to liberate the dye. Thus, reagent costs and other difficulties of conventional kinetic tests for measuring amylase are eliminated, namely the initial phase of non-linear kinetics, which typically lasts several minutes. On the other hand, when a new substance is used, the dynamics proceed linearly from the beginning. This allows considerably faster measurements by hand and above all by automatic analyzers.

Using 3-0-(α-D-maltotriosyl)-5-hydroxy-2-nitropyridine (compound 812), the current state of the art also works without the involvement of the coenzyme α-glucosidase and immediately in linear motion. A sufficiently high absorption change was achieved compared to the standard.

Using 3-0-(α-D-galactopyranosyl)-3-hydroxy-2-nitropyridine, the activity of α-galactosidase in human urine could be measured kinetically. For this purpose, add a 10-fold concentrate of normal urine 0,2- in a buffered substrate solution 1- and 405 nm and 2
The absorbance change at 2°C was recorded. 0.0 per minute under these conditions
The absorbance difference of D12A was measured. This substrate is not degraded by β-galactosidase.

β-galactosidase activity is 2H-7-0-(β-D-
Galactopyranosyl)-4-methyl-8-nitro-benzobilan-2-one (compound 515) could be used to determine kinetics in human urine. 200g buffered substrate solution
and absorbance changes of Q, 014 A per minute were recorded at 23° C. and 366 nm using 200 μt of urine of normal concentration. This substrate is not degraded by α-galactosidase. β-galactosidase in human urine could also be measured kinetically when resorufin-β-D-galactopyranoside (compound S6) was used. Here, 37℃, 5
An absorption change of 0.0038 A per minute was achieved at 78 nm.

The activity of β-glucuronidase in human urine is 2H
-7-0-(β-D-glucuronopyranosyl)-4-methyl-8-nitrobenzopyran-2-one (compound 89
) was able to be measured dynamically. 0.1 ml of the urea concentrate was then added to 0.6-ml of the buffered substrate solution and the activity was recorded at p)T5.30°C and 366 nm.

Under these conditions, an absorption difference of 0.014 A per minute was measured.

This measurement is therefore many times more sensitive than using the conventional p-nitrophenyl substrate (see Table 4).

Table 4 Determination of β-glucuronidase in human urine at pH 5 Compounds s9 0.014 p-nitrophenyl substrate 0.0005 Substrate p using N-acetyl-β-glucosaminitase from bovine kidney
-nitrophenyl-N-acetylamino-glucoside and 2H-7-0-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-4-methyl-8-nitrobenzopyran-2-one (compound BB ) were compared with each other. Enzyme solution diluted 1:101 with 0.5 mg of substrate solution (Sigma product) 0.1
tnt and 22 °C and 405 nm or 3
Absorption changes were measured at 65 nm. The results are summarized in Table 5.

The absorption change is p for E8 when adjusted to pH 4 or 5.
-Nitrophenylacetylamino-glucoside about 6
6 times thicker.

Activity in human urine (10-fold concentrated) can also be measured kinetically using S8. Absorption change per minute at pH 5.5 measured at 57° C. and 365 nm using 0.5 − of buffered substrate solution and 0.1 − of 10-fold concentrated normal urine.
D 23 A and 0.014 A per minute were obtained at pH 4,s. This change in absorbance was so large that the activity could be measured kinetically with sufficient sensitivity even in the same sample volume of undiluted urine.

Table 5 Measurements of N-acetylglucosaminider 7 p-nitrophenyl substrate 0.0076 compound s3 0.266 2H-7-0-4-methyl-8-nitro-benzopyran-2-one using phospade (compound s 13) The activity of acid phosphatase could be measured kinetically with considerably more sensitivity than using nitrophenylphospade. Measurement is 1
Tests were performed using both substrates in semen-plasma diluted to 1:101. 1 s/! of buffered substrate solution at pH 4.8!
Add 0.01 m of the diluted sample to
Kinetic measurements were taken at 05 or 365 nm.

As shown in Table 6, the absorption change is approximately 15 times greater with s15 than with nitrophenylphospade.

Table 6 Compounds s13 0.0975 15 p-Nitrophenylphospade 0.0075 1 The invention also relates to absorbent carriers, film layers, loose or compact powder mixtures, formula A-0-B (wherein A-OH is a dye with the general formula ■ or ■ and BOH is an amino acid or an oligoR peptide with a protecting group if necessary) and customary additive substances such as buffer substances, washes. The present invention also relates to a diagnostic agent for the detection of esterases, especially esterases present in leukocytes, consisting of a lyophilized composition or solution containing an agent, an activator or an inhibitor or/and a stabilizer.

Furthermore, the present invention also relates to the use of a compound having the formula A-0-B as described in the previous section for the preparation of a diagnostic agent for detecting esterases present in body fluids, in particular esterases present in leukocytes.

Substances present at very low concentrations should generally be detected by enzyme immunoassay. The sensitivity of this detection system is thus of great importance.

Significant increases in sensitivity could be achieved in current state of the art enzyme immunoassays when using the new substrate. In this assay, g'B concentrations are measured by indirect antibody techniques as a measure of allergy.

Approximately 4.3 times higher absorption and 5-hydroxy- About 1.7 times higher absorption could be measured with 2-nitropyridine-β-galactoside.

Therefore, for resorufin-β-galactoside, the incubation time for the enzymatic reaction can be considerably shortened for the same sensitivity, or the sensitivity of the gB detection can be greatly increased for the same incubation time. These two steps can be of great practical importance. Whether greater sensitivity or faster test results are important depends on the substance to be detected.

Table 7 Resorufin-β-galactoside 576nm 0.7
610-nitrophenylgalactoside 405 nm
The following examples are provided to illustrate the present invention.

Example 1 2H-7-0-(α-D-glucopyranosyl)-4-methyl-8-nitrobenzopyran-2-one (compound 5i
) 1-β-chloro-3,4,6-triacetylglucopyranoside 1731t”) 2H-7-hydroxy- in toluene
4-Methyl-8-nitropensohylan-2-one 15
The mixture was heated under reflux for 9 and 20 hours with exclusion of water. The solvent was then evaporated and the residue was taken up in 0H20p2, washed with saturated NaHO05 solution, dried and concentrated again. The syrupy material was dissolved in hot methanol and crystallized. Yield 22.59C (75% of theory).

Crystals of conotriacetyl derivative in 200ml of methanol/triethylamine/water (50:5:5) mixture!
It became cloudy and was stirred and it dissolved after about 40 minutes.After 1-2 hours the deacetylated glucoside separated and it was left overnight at 0 DEG C. and the precipitate was filtered and washed with methanol. Further material was obtained by repeated precipitation from the mother liquor. Total yield 11a (theoretical amount 6
5%), melting point 130°C.

Elemental analysis value (molecular formula C16H1701ONs molecular weight 38
3.315) 0% H-chi N-chi 0% Calculated value: 50.1 4.5 5.7 41.7 Actual value: 49.9 4.5 3.7 41.7 Example 2 3-0- (α-D-gelcopi2-nosyl)-2-nitropyridine (compound 82) Compound S2 is 2H-7-hydroxy-4-methyl-8-
Nitropensohyran-2-one to 5-hydroxy-2
-Prepared similarly to compound S1 except for substitution with iop-nitropyridine. Melting point: 178°C.

Elemental analysis value 0% H% 8% 0% Calculated value: 43.7 4.7 9.4 42.2 Actual value: 43.7 4.8 9.1 42.5 Example 6 3-0-(α -D-G2-nitropyridine (compound 83) An intimate mixture of acetobromogalactose 419 (0,1 mol) and 3-hydroxy-2-nitropyridine 28F (0,2 mol) with moisture blocking The mixture was melted by heating to 70° C. with thorough stirring. Hg (ON)
After adding the mixture of 2139 and HgBr21P, the temperature was brought to 100°C with stirring (1 hour) (HON
occurrence). After cooling, 50% ethyl acetate was added to aaβ42001.
11fi was added, treated with activated carbon 19 and then filtered through silica gel 10jI. The Oki solution was shaken with saturated bicarbonate and then with jN KBr solution, dried and evaporated to dryness. The syrupy residue was dissolved in hot methanol and crystallized at 0°C.

Crystals of the tetraacetyl compound were suspended in 200 g of a methanol/triethylamine/water (50:5:5) mixture and stirred, which dissolved after about 40 minutes.

After 1-2 hours the deacetylated α-galactoside was separated. This was left at -0°C overnight, filtered and washed with methanol. Melting point: 220°C.

Elemental analysis value (molecular formula 〇1 jH1408N2, molecule: 1
Calculated value: 43.7 4.7 9.2 42.3 Actual value:
43.8 4.8 9.2 42.8 Example 4 2H-7-0-(β-D-glucopyranosyl-4-y
'f-8-nitrobenzobilan-2-one (compound 8
4) 2H-7-hydroxy-4-methyl-8-nitrobenzopyran-2-one 189. acetobrome-glucose 1
6jl and anhydrous Na2003 50p in acetone 20
Boil under reflux in 0 ml for 20 hours, filter the mixture, concentrate and take up the residue in chloroform i), C1,I
Washed with N NaOH, dried, concentrated again and the residue was then taken up in methanol to crystallize the tetraacetyl derivative. For deacetylation, the tetraacetyl derivative was stirred in methanol with a catalytic amount of sodium methylate for about 8 hours. Melting point: 210°C.

Elemental analysis value 0chi Hchi N% Ochi calculated value: 50.1 4.5 3.7 41. ．． 7 Actual value: 50.4 4.6 3.7 41.2 Example 5 3-○-(β-D-glucopyranosyl)-2-nitropyridine (Compound 85) Compound S5 is 2H-7-hydroxy-4- Methyl-8-
Nitrobensohyran-2-onwo-3-hydroxy-2-
It was prepared in the same manner as Compound S4 with multiple substitutions at am 162 of ditropyridine. Melting point: 206°C.

Elemental analysis value: ±(Dan1-Lee calculated value: 43.7 4.6 9.2 42.3 Actual value:
43.5 4.5 9.1 42.3 Example 6 Resocinin-8-β-D-galactopyranoside (Compound 86) Resazurin-Na 5 jl was stirred with acetobromogacitose 8.29 in acetonitrile for 5 days. did.

The solvent was removed and the residue was taken up in ethyl acetate and shaken with saturated NaHO05 solution. After drying, the ethyl acetate was also removed under vacuum and the residue consisting of resazurin-β-galactoside tetraacetate was crystallized from methanol.

Resorufin-β-D-galactopi-dinoside is resazurin-β-D-galactopyranoside tetraacetate:・
(by hydrogenation in methanol using palladium/activated carbon as catalyst).

If the solution needed to be completely colorless, 1 vol of a solution of 0.1N sodium methylate in 100 ml of the solution was added, stirred for 6 hours and filtered through silica gel 109. When the mixture was concentrated while blowing air, resolin galactoside crystallized. After 24 hours at 0° C., the orange crystals were filtered off with suction. Melting point: 222°C.

Elemental analysis value 0% H% 8% 0% Calculated value: 57.6 4.6 3.7 34.1 Actual value:
57.8 4.9 3,4 34.2 Example 7 3-0-(β-D-galactopyranosyl)-2-nitropyridine (Compound S7) Compound S7 is prepared in the same manner as Compound S4, but , except that 2H-7-hydroxy-4-methyl-8-nitrobenzopyran-2-one is polysubstituted with silver salt 169 of 3-hydroxy-2-ditropyridine, and acetobromoglucose is replaced with acetobromogalactose. Multiple substitutions were made. Melting point: 245°C.

Elemental analysis value 0% Hchi 8% Ochi calculated value: 43.7 4.7 9.4 42.2 Actual value: 43.7 4.6 9,342.15I! Example 8 2H-7-〇-(2-acetamido-2-deoxy-β
-D-glucopyranosyl)-4-methyl-8-nitrobenzopyran-2-one (2H-7-hydroxy-4-methyl-8- Acetone 200I with nitrobenzopyran-2-one 6.659
Na0H2jl dissolved in nIl and in a small amount of water
After the addition of the solution, the mixture was stirred at room temperature for 1 day and kept at 0-5°C for an additional 5 days.

The acetone was removed under reduced pressure, the residue was taken up in 0HOIIs, shaken with saturated NaHCO3 solution, dried and the OHOIIs was removed. The residue crystallized immediately upon treatment with warm methanol. Solution 10 for deacetylation
A solution of 0.1 N sodium methylate in 0.0 ml of dimethateol was used. Melting point 216-218°C.

Elemental analysis value: L ±(Tama) Calculated value: 50.9 4.8 6.6 37.7 Actual value:
50.14.9 6.5 38.1 Example 9 2H-7-0-(1-β-D-glucuronopyranosyl)
-4-methyl-8-nitro-benzopyran-2-one (
Compound S9) Fifteen portions of compound S4 in a 1.5β stirred flask equipped with a high-performance stirrer were dissolved in phosphate buffer 600, pH 6.8, at room temperature in the presence of platinum black 29 by bubbling oxygen gas. Schiff oxidized for 2 hours. After warming at the end and filtering off the catalyst with suction, solution F was acidified, evaporated to dryness and the residue was extracted with hot ethyl acetate. When left in water, the desired β
- Glucuronide crystallized.

Elemental analysis value 0% H% 8% Ochi calculated value: 46.2 4.1 3.4 46.2 Actual value: 45.1 3.9 3,2 44.6 Example 10 2H-7-0 -(α-D-maltotriosyl)-4-)
f-8-nitrobensohylan-2-one (compound 51
0) Compound S119 and α-cyclodextrin 5p together with cyclodextrin-glucosyl-transferase in morpholine-ethanesulfonic acid buffer pH 6,
After culturing for 2 to 5 days in Bio-Rad P2 (4
Chromatography was carried out on a 150×50 column at 3-5 bar, 30° C. and a flow rate of 120-250 m/h. The eluent was degassed water.

The various 7-lactones were collected and lyophilized.

Elemental analysis value 0% I (% N4 0% Calculated value: 45.5 5.5 1.8 47.3 Actual value: 45.5 5.3 1.9 44.5 Example 11 2H-7-0- (α-D-maltotetraosyl)-4-
Methyl-8-nitrobenzopin-2-one (compound 5
11) Compound 811 was prepared similarly to compound S10, but the 75 fractions eluted from the chromatography column after compound S10 were collected and lyophilized.

Phenyl column manufactured by HPLO Analytical Water Company (Fa, Watθr) [Bonda, pak-P
henyl).

10μm, 3.9X! +00 simple] Clean water/methanol (7
0:3Q) mixture and a flow rate of 1.12 m/min.

Compound 811 is eluted under these conditions after an elution time of Z3 minutes.

Example 12 3-0-(α-D-maltotriosyl)-2-nitropyridine (Compound 512) Compound 812 is prepared similarly to Compound 810, except that Compound S1 is replaced by Compound 1p. S2 was used.

Elemental analysis value Cchi H% Qchi N% Calculated value: 41.6 5,7 48.3 4.2 Actual value:
41.4 5.6 46.8 3.1 Example 13 2H-7-0-(phosphoryl) to 4-methyl-8-nitrobenzopyran-2-one (compound S13) All reagents must be carefully dried .2H-
12.5 jJ of 7-hydroxy-4-methyl-8-nitrobenzopyran-2-one were dissolved in 200 ml of pyridine. This solution was mixed with POO et al.
It was added dropwise to the solution of No. 2 while stirring. At that time, care was taken to ensure that the temperature did not exceed 5°C. After 30 minutes the reaction product was decomposed (by addition of ice water (100 ml)). Next K 1 ON
This solution was adjusted to pH by adding NaOH dropwise under stirring.
Adjusted to 7.5.

Pyridine and water under reduced pressure at the lowest possible temperature (50℃)
evaporated with. To the residue was added 30 mQ of acetone, mash and extracted with 125 mQ of warm 80 volume diluted methanol. 600 μm of acetone was added to the methanol solution. Compound 813 was obtained as the disodium salt.

Recrystallized from 70 volumes of aqueous ethyl alcohol.

Example 14 Resolinphospade (compound S 14 ) Sodium resazurin 2.5 in dry acetonitrile 501n1
p (0,o 1 mol of acetonitrile 50111
A solution of 4.5 P of bis-2,2,2-trichloroethyl-phosphoryl-chloride in 1 was added dropwise to the solution. When the addition was complete, 5 g of pyridine was added. The mixture was evaporated under reduced pressure, the residue was taken up in chloroform, washed with NaHOO5, dried, concentrated and the residue was crystallized from ethanol. The crystallized product was obtained by reduction with pyridine/zinc dust, during which the preservative Mi& was also removed.

Example 15 3H-7-Hydroxy-9-cyanoxanthin-6-one (Cyanorufin, Compound 01) 3H-7-Hydroxy-9-hydroxyiminomethyl-xanthine-3-one 9F and in anhydrous dioxane To a water-cooled, stirred solution of 11.5 mm of anhydrous pyridine was added dropwise 5.5 mm of trifluoroacetic anhydride at a rate such that the temperature was maintained below 5°C. After the addition, the mixture was brought to room temperature and stirred for a further 6 hours, with ice-cooled 2NHO1
200 Iffl and the separated precipitate was vacuumed, washed with water and dried. Recrystallized from ethanol, yield Z72 (89.5% of theory).

The melting point of cyanorphine was 300°C or higher.

Cyanorphine has a pKa value of 5.55 with a maximum absorption at 578 nm and a molar absorptivity of 64,42017 moles/cns at 578 nm and pH 71.

Elemental analysis 4a (molecular formula 014H705N 1 molecule 1i
237217) (J H% 0% N% Calculated value: 70.9 5.0 20.7 5.9 Actual value: 69.6 3.6 20.7 5.3 Advantages of this new chromogen is due to its low pKa value, its very high extinction coefficient and its absorption in the long wavelength range.

Example 16 Preparation of N-1 luenesulfonyl-L-alanine (tosylalanine) 0.01 mole of -1-alania is dissolved in 20 ml of 1 molar sodium hydroxide solution and added to tD in diethyl ether 10 + d. A 0.01 molar p-toluenesulfonic acid chloride solution was added and the mixture was stirred vigorously for t-4 hours. The ether phase was separated and the aqueous phase was acidified to pH 3 using concentrated hydrochloric acid. The crude N-)luenesulfonyl-L-anine began to crystallize (cooled to 4 DEG C. if necessary) and was recrystallized from 60 ml of aqueous alcohol after treatment. Melting point: 150-131°C.

Example 17 Preparation of tosylalanylresorufin Dicyclohexylcarbodiimide 220!1f (1,1 mmol & 1-hydroxybenzotriazole 165~) in dimethylformamide (DMF) Sme
(1.1 mmol) and 1 mmol of tosylalanine were dissolved at room temperature under stirring. Next, add this to resorufin [
(product of Alclrich, USA) 210
~(1 mmol) was added and stirred for an additional 2 hours.

This mixture was then poured into 100 ml of ice water. Add in and ethyl acetate: i! Shake with 5Qmil. After separation, the ethyl acetate phase was dried over sodium sulfate and the ethyl acetate was removed under reduced pressure.

Product purified by 2-layer chromatography [2-layer: silica gel, mobile phase: 0HOA5+0H50H (91)]
had a melting point of 165°C and was pure on a thin layer chroma dog.

Example 18 Toll-Gly-Pro-Arg-Resazurin v4
1 mmol of Tos-Gly-Pro-Arg-OH
(dry) was suspended in 30ml of tetrahydrofuran (dry). To this was added 4 mmol of chloroformic acid ethyl ester, the mixture was stirred for about 15 minutes and cooled to -10°C. 3 mmol of N-methylmorpholine was added and stirred for 60 minutes at room temperature.

To this was added dropwise 3 mmol of resazurin in dry cotetrahydrone 6OIIL. The reaction was complete after 4 hours, the F solution was evaporated and the crude product was transferred to a chromatogelan e-column [stationary phase Purified with Nijiri gel, mobile phase: OHC!β3+glacial acetic acid (19:1).

A test strip is impregnated with a 0.1 thialcohol solution of this substrate and, when dried, immediately produces a violet color when moistened with a disintegrating solution.

Example 19 Determination of α-Amylase Activity Using Compound 810
0A 585”l and compound S 1020011jl
was dissolved. Two volumes of urine (2QmQ) were added to one volume of this solution using a piston and mixed well. Absorption changes were continuously recorded using a photometer equipped with a 365-nun photometer.

Based on the record of the pen recorder, the absorption change (δ
A/min) and use the formula below to calculate the enzyme activity (U
/l) was calculated.

102 - Test volume containing sample 0.02 - Sample volume 1 to 1000 - Conversion factor from 16.1 to L 16.1 - 2H at pH 6,0 and 365 nm
Absorbance of -7-hydroxy-4-methyl-8-nitrobenzopyran-2-one an2/μmol Other hydro-2-ases are prepared according to the invention and under adapted reaction conditions using substrates specific for the enzyme. It can be measured in the same manner below.

Example 20 Determination of leukocyte esterase using tosyl-alanyl-resorufin Indicator test strips No. 218 from Maaherey-Nagel, Diren, Federal Republic of Germany, were impregnated sequentially with the following solutions and dried. .

a) 0.25 mol/aqueous borate buffer with pH 8.

b) Tosyl-0.259 dissolved in 1 L of ethyl acetate
Alanyl-resorufin.

A drying temperature of approximately 80° C. for solution a) and approximately 120° C. for b) was chosen.

Suspensions with various white blood cell concentrations were used as samples, into which test strips were briefly immersed. A reddish-purple color appears after about 1 minute, the intensity of which depends on the white blood cell concentration.

The detection sensitivity of this test system is 1000 white blood cells/μp.

By using N-tosyl-aminobutyryl-resorufin or Boa-Leu-resorufin in the impregnating solution b), test strips were obtained whose detection sensitivity was 1000 leukocytes and 20[]0/μβ, respectively.

Example 21 2H-7-0-(β-G2Ctopi2nocylin-4-methyl-8-nitrobensohylan-2-one (Compound 515
) Preparation Compound 815 was vsm similarly to compound S4, except that acetobromogalactose 16jI was used in place of acetobromoglucose. Melting point 188-192
℃.

Elemental analysis value C H-N Chi O-chi Calculated value: 50.1 4.5 3.7 41.7 Actual value: 50.2 4.6 3.7 413 Patent applicant Behringwerke Akchengesellschaft outside Continuation of page 1 for 2 people

Claims (1)

[Scope of Claims] 1] A compound having the formula, where A is a heterocyclic compound A-OH having an acidic pKa value whose absorbance after hydrolysis of A-0-B is measured photometrically. represents the residue of the ion A-0'', while B is the residue of the compound B-OH, which makes that compound specific for reaction with certain enzymes, and in which case A-OHQ to a0 formula I R (in the formula, R1 is OH, NO2 or halogen, and R5,
R4 and R5 can be H or NO2, with up to 2 NO2 being present], b0 compounds having the formula 3 (wherein R1 is H or 1 to 12, preferably 1 to 3 carbons); Alkyl with atoms and R2, R3 and R4
can be H or NO2, in which case 1 or 2 NO2 are present);
CH3)2 and I (3 can be O or S), or d0 compounds having the formula ■ (wherein R1 is aryl, preferably phenyl, CN or 1 to 12 preferably 1 to 3 carbon atoms) ] and B-OH is a sugar or sugar derivative, especially glucose, maltodextrin, galactose, glucuronic acid, N-acetylglucosamine or fucose, an amino acid or an oligonucleotide (these are in particular N-)dylalanine or CBZ-glycine, each of which may carry a protecting group, in particular a toluenesulfonyl group or a carbobenzyloxy (CBZ) group, or an inorganic acid, in particular phosphoric acid or sulfuric acid. 2) Formula A- A process for preparing a compound having the formula A-0-B according to claim 1, which comprises reacting a compound having an OH with a reactive conductor of a compound having the formula B-OH. 3) Use of a compound having the formula A-0-B according to claim 1 as a substrate for the detection and measurement of hydrolases. 4) Absorbent carrier, film layer, loose or compact powder mixture, formula A-0-B, where A-OH is a dye having formula ■ or ■ and BOH is provided with protecting groups if necessary. amino acids or oligos) and customary additive substances such as buffer substances, detergents,
A diagnostic agent for the detection of esterases, in particular esterases present in leukocytes, consisting of a lyophilizate or a solution containing an activator or an inhibitor or/and a stabilizer.